Nucleic acid analysis has played an important role for the detection of pathogens and genetic diseases. In recent years, its usefulness has been seen in many decentralized applications such as point-of-care diagnostics, environmental and food monitoring, and the detection of biological warfare agents. Among the available analytical techniques for DNA analyses, real-time polymerase chain reaction has been a key technology for high-speed testing and accurate quantification.
Various assays based on real-time PCR have been developed utilizing fluorescence-linked reporters such as SYBR Green 1, hydrolysis probe, and hybridization probes for simultaneous deoxyribonucleic acid (DNA) amplification and PCR amplicon detection. Despite wide acceptance, their use is largely limited in clinical and research laboratory settings. The difficulty in advancing this technology for point-of-care testing (POCT) applications lies in the requirement of bulky and complex optical systems for the DNA amplicon detection. The goal of performing complete DNA analyses with a hand-held instrument is not attainable based on optical detection systems that are bulky and cumbersome. A far more suitable alternative for this type of use and one that is extremely suited for POCT, is a system based on the detection of electrochemical signals.
Over the past years, numerous studies have been carried out on electrochemical DNA sensors, some of which focused on PCR amplicon detection. Efforts have also been made in developing DNA microchips having an attached electrochemical signaling label employed in conjunction with an electrochemical detection system for post-PCR product identification. The latter prior art post-PCR hybridization-based platform suffers from a long assay time and has a narrow dynamic range when compared to fluorescence-based real-time PCR methods.
In view thereof, there is a need for developing a method for detecting and quantifying nucleic acid(s) in a sample that is accurate, reproducible, and safe and, at the same time, may be performed in small scale devices.